efficacy of the field controls skye unit against

Innovative Bioanalysis, Inc. FIELD CONTROLS SKYE/AEROSOL SARS-COV-2 of 14

EFFICACY OF THE FIELD CONTROLS SKYE UNIT AGAINST AEROSOLIZED

SARS-CoV-2 USA-CA1/2020

PROJECT: FIELD CONTROLS EFFICACY ON AEROLIZED SARS-COV-2

PRODUCT: AIR+HEALTH SKYE

CAP LIC NO: 8860298

CLIA LIC NO: 05D0955926

STATE ID: CLF 00324630

CHALLENGE ORGANISM(S):

SARS-CoV-2 USA-CA1/2020

Dana Yee, M.D.

Medical Director

Testing Facility

Innovative Bioanalysis, Inc.

3188 Airway Ave Suite D

Costa Mesa, CA 92626

www.InnovativeBioanalysis.com

Email: [email protected]

Laboratory Project Number

1082

DocuSign Envelope ID: CD80ED40-DE78-4C37-A0ED-800AB5FD82E2


Table of Contents EFFICACY OF FIELD CONTROLS SKYE AGAINST AEROSOLIZED SARS-CoV-2 USA-CA1/2020 .................................... 1

Efficacy Study Summary ....................................................................................................................................... 3

Study Report ........................................................................................................................................................ 4

Study Title: ....................................................................................................................................................... 4

Sponsor: ........................................................................................................................................................... 4

Test Facility: ..................................................................................................................................................... 4

Device Testing: ................................................................................................................................................. 4

Study Report Date: 6/21/2021 ......................................................................................................................... 4

Experimental Start Date: 05/11/2021.............................................................................................................. 4

Experimental End Date: 05/31/2021 .............................................................................................................. 4

Study Completion Date: 06/21/2021 ............................................................................................................... 4

Study Objective: ............................................................................................................................................... 4

Test Method: .................................................................................................................................................... 4

Test System Strains: ......................................................................................................................................... 5

Study Materials and Equipment: ..................................................................................................................... 5

Test Method: .................................................................................................................................................... 8

Protocol Changes: .......................................................................................................................................... 11

Control Protocol ............................................................................................................................................. 11

Study Results ...................................................................................................................................................... 12

Conclusion: ......................................................................................................................................................... 13

Disclaimer........................................................................................................................................................... 14



Efficacy Study Summary

Study Title EFFICACY OF FIELD CONTROLS SKYE AGAINST AEROSOLIZED SARS-COV-2 USA-

CA1/2020

Laboratory Project # 1082

Guideline: Modified ISO standards as no international standards exist.

Testing Facility Innovative Bioanalysis, Inc.

Study Initiation Date: 05/11/2021

Study Completion Date: 05/31/2021

GLP Compliance All internal SOPs and processes follow GCLP guidelines and recommendations.

Test Substance SARS-CoV-2 USA-CA1/2020

Description The Field Controls SKYE system is a portable air purifier designed to be placed

in a free-standing room to decrease the concentration of pathogens in the air

when operating. This in vitro study is being conducted to determine the

effectivity of the SKYE unit in reducing an aerosolized pathogen, SARS-CoV-2

when operating.

Test Conditions The test was conducted in a large, sealed environment that complied to BSL-3

standards and was inspected for any leaks prior to usage. The temperature

during all test runs was approximately 75F ±2F, with a relative humidity of

36%. Air samplers were calibrated by the manufacturer on September 3, 2020

and set at a standard flow of 5.02L/min. Calibration records indicate a 0.20%

tolerance. The nebulizer was filled with the same amount of viral stock (6.32 x

106 TCID50 per mL) in FBS-based viral media and nebulized at a constant rate

while four mixing fans were running simultaneously to ensure homogenous air.

Test Results When tested against SARS-CoV-2 USA-CA1/2020 virus, the SKYE unit showed a

reduction of active pathogens after 30 minutes of continued operation

resulting in a loss greater than 1.20 x 102 TCDI50/mL.

Control Results A control test was conducted without the air purifying unit engaged and

samples were taken at the corresponding timepoints used for the challenge

trial to serve as a comparative baseline in order to calculate viral reduction.

Conclusion The SKYE unit demonstrated a 99.99% net reduction of recoverable active

SARS-CoV-2 virus in the air of after 30 minutes of operation.



Study Report

Study Title: FIELD CONTROLS SKYE AGAINST AEROSOLIZED SARS-COV-2-USA-CA1/2020

Sponsor: Field Controls, LLC

Test Facility: Innovative Bioanalysis, Inc. 3188 Airway Ave Suite D, Costa Mesa, CA 92626

Device Testing: Testing the efficacy of the SKYE unit against an aerosolized known pathogen, SARS-CoV-2

Study Report Date: 6/21/2021

Experimental Start Date: 05/11/2021

Experimental End Date: 05/31/2021

Study Completion Date: 06/21/2021

Study Objective:

This in vitro study was designed to determine the efficacy of the SKYE unit against the airborne transmission of

the known pathogen, SARS-CoV-2.

Test Method:

Bioaerosol Generation:

For the control and the viral challenges, the nebulizer was filled with the same amount of viral stock (6.32 x 106

TCID50 per mL) and nebulized at a flow rate of 1mL/min. The nebulizer was driven by untreated local

atmospheric air. After each completion, the nebulizer's remaining viral stock volume was weighed to confirm

that the same amount of viral stock was nebulized.

Bioaerosol Sampling:

For air sampling, 2 different Gilian 10i programmable vacuum devices were used. Air samplers were calibrated

by the manufacturer in September 2020 and certificates were inspected prior to use. Air sample volume

collections were confirmed prior to use with a Gilian Gilibrator 2, SN- 200700-12 and a high flow bubble

generator SN-2009012-H. Air samplers were operated in conjunction with removable sealed cassettes, which

were manually removed after each sampling time point. Cassettes had a delicate internal filtration disc to

collect viral samples which were coated with a viral suspension media to aid collections.

Aerosolization of Viral Media:

Control samples were performed in the same manner as the viral test at the timepoints and rate of collection.

A viral stock of SAS-CoV-2 USA-CA1/2020 with a concentration of 6.32 x 106 TCDI50/mL was used for this

experiment.



Test System Strains: SARS-CoV-2

The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through

BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-CA1/2020, NR-52382.

Study Materials and Equipment:

Equipment Overview: The equipment arrived at the laboratory

pre-packaged from the manufacturer and was inspected for

damage upon arrival. All filtration systems were installed prior to

arrival at the laboratory. The device was powered on to confirm

functionality prior to testing.

MANUFACTURER: Air Health

MODEL: SKYE

SIZE: 26” X 11.4” X 11.3”

MAKE: Air+Health

SERIAL #: N/A

Equipment Specifics: The equipment arrived at the laboratory pre-packaged from the manufacturer and was

inspected for damage upon arrival. The system has an internal fan system along with a multistage mechanical

filtrations system.

Testing Chamber: The test was conducted inside a 10’ X 8’ X 8’, sealed chamber with active monitoring of

testing conditions via calibrated wireless devices and air sampling sensor. At each corner of the chamber, low

volume mixing fans were positioned to ensure homogenous, bioaerosol concentrations. Furthermore, the use

of the mixing fans encouraged bioaerosol suspension and reduction in natural particle descent rates. The

testing chamber was set up to allow all exhausted air after the test samples had been taken to be exhausted

through a dual HEPA filtration system.



Design Layout:

The chamber used for testing was a 10’ X 8’ X 8’, sealed air volume chamber with metal walls and epoxy floor

which complied with BSL3 standards. The chamber was designed to be completely sealed from the outside

environment to prevent any potential release of testing media into the atmosphere. The testing chamber was

equipped with 2 sealed viewing windows and a lockable antechamber for entry and exit. Calibrated wireless

devices and air sampling sensors are strategically placed to provide active monitoring of testing conditions –

temperature and humidity—throughout the testing process.

At each corner of the chamber, low volume mixing fans were positioned at 45-degree angles to ensure

homogenous mixing of bioaerosol concentrations when nebulized into the chamber. Furthermore, the use of

the mixing fans encouraged bioaerosol suspension and reduction in natural particle descent rates. The fans

help circulate the air around the room, allowing a mixing of the nebulized viral media and air within the testing

chamber. The testing chamber was set up to allow all exhausted air after the test samples had been taken to be

passed through a dual HEPA filtration system. The testing chamber had HEPA filtered inlets and exhaust,

coupled with an active UV-C system in all ducting lines.

For air sample testing, the chamber was equipped with 2 probes that were along the centerline of the room

and protruded down from the ceiling 24”. Each probe was connected to a Gilian 10i programmable system

with sampling cassettes from lot #24320 made by Zefon International. A single bioaerosol nebulizing port was

in the center of the 10’ wall opposite of the entry doors. The dissemination port protruded from the wall 24”

and was connected to a programmable compressor nebulizer system.

An active air sampling sensor was used to confirm operations of the equipment and O3 measurements were

taken only for verification the system was operating. Test scenario captures O3 data, but the conditions are not

designed to be compared to EPA requirements and cannot be used for O3 claims as the sensors and test

parameters are not designed to meet O3 certification requirements. The bioaerosol testing system was

constructed to meet internal SOP requirements and all seams were sealed. During testing, the temperature was

75°F with a relative humidity of 36%. All sample collection pumps were set to a 10-minute air draw at the point

of sampling.

Prior to testing, the chamber was pressure tested for leaks and visual inspections were made using a colored

smoking device. All seals for the chamber were confirmed and all equipment used had a function tests to

confirm working conditions. For calibrated equipment, calibration records were checked to confirm

operational status.



Figure 1. Room layout for control and experimental trial.



Test Method:

Field Controls supplied the SKYE air purifier system for testing purposes to determine the efficacy against

aerosolized viral pathogens. More specifically, this study evaluated the device’s ability to inactivate the viral

strain referred to as SARS-CoV-2 in the air in a defined enclosed space.

Exposure Conditions:

1. Prior to the initial control test and following each trial run, the testing area was decontaminated and

prepped per internal procedures.

2. The temperature during all test runs was approximately 75F with a relative humidity of 36%.

3. Air samplers were calibrated by the manufacturer on September 3, 2020 and set at a standard flow of

5.02L/min. Calibration records indicate a 0.20% tolerance.

4. The air sample collection volumes were set to 10-minute continual draws at the point of sampling.

5. Low volume mixing fans were placed at each corner of the chamber at a 45-degree angle and turned on

prior to nebulization.

6. Each timepoint was treated as an individual test and the chamber was reset after sample collection.

7. Testing timepoints were as follows with T equal to minutes: T-0, T-15, T-30

Nebulization:

1. Nebulization for control and viral test challenges were performed in the same manner.

2. After nebulization of the pathogen, the SKYE unit was turned on with a remote power switch.

3. Fan speed on the SKYE was set on high for the test conditions and was turned off at the defined

timepoint for sample collection.

4. For the viral challenge, a known quantity of viral media was nebulized into the sealed environment

from a dissemination port.

5. Viral media was nebulized at a constant rate for 12.5 minutes.

6. Air sampling collection occurred for both the challenge and control tests at the defined sample

timepoints after nebulization ceased for a total of 10 minutes.

7. Sample cassettes were manually removed from the collection system after each control run and each

air pass challenge.

8. Upon cassette removal after each challenge, cassette sets were taken to an adjacent biosafety cabinet

for extraction and placement into viral suspension media.

9. One control and viral challenge was conducted using the same methodology.



Post Decontamination:

At the conclusion of each viral challenge test the UV-C system inside the testing chamber was activated for 30

minutes. After 30 minutes of UV-C exposure the chamber was fogged with a Hydrogen Peroxide gas mixture

followed by a 30-minute air purge. All test equipment was cleaned at the end of each day with a 70% alcohol

solution. Collection lines were soaked in a bleach bath mixture for 30 minutes then rinsed repeatedly with DI

water. Nebulizer and vacuum collection pumps were decontaminated with Hydrogen Peroxide mixtures.

Preparation of The Pathogen

Viral Stock: SARS-CoV-2 USA-CA1/2020 (BEI NR-52382)

Test Specifications Results

Identification by Infectivity in Vero 6 cells

Cell Rounding and Detachment Cell Rounding and Detachment

Next-Generation Sequencing (NGS) of the complete genome using Illumina® iSeq™ 100 Platform

≥ 98% identity with SARS-CoV 2, isolate USA-CA1/2020 GenBank: MN994467.1

99.9% identity with SARS-CoV 2, isolate USA-CA1/2020 GenBank: MN994467.1

Approx. 940 Nucleotides ≥ 98% identity with SARS-CoV 2, strain FDAARGOS_983 isolate USA-CA1/2020 GenBank: MT246667.1

100% identity with SARS-CoV 2, strain FDAARGOS_983 isolate USA-CA1/2020 GenBank: MT246667.1

Titer by TCID50 in Vero E6 Cells by cytopathic effect

Report Results 2.8 X 105 TCID50 per mL in 5 days at 37°C and 5% CO2

Sterility (21-Day Incubation) Harpos HTYE Broth, aerobic

No Growth

No Growth

Trypticase Soy Broth, aerobic

No Growth

No Growth

Sabourad Broth, aerobic

No Growth

No Growth

Sheep Blood Agar, aerobic

No Growth

No Growth

Sheep Blood Agar, anaerobic

No Growth

No Growth

Thioglycollate Broth, anaerobic

No Growth

No Growth

DMEM with 10% FBS No Growth

No Growth



Mycoplasma Contamination Agar and Broth Culture

None Detected

None Detected

DNA Detection by PCR of extracted test article nucleic acid

None Detected None Detected

*The viral titer listed in the Certificate of Analysis is representative of the titer provided by BEI Resources. These viruses

are grown on VeroE6 cells either in-house or at a partner lab to the concentrations listed within the experiment design.

TCID50 Procedure:

Materials and Equipment:

• Certified Biological Safety Cabinet

• Micropipette and sterile disposable aerosol resistant tips—20uL, 200 uL, 1000uL

• Inverted Microscope

• Tubes for dilution

• Hemocytometer with coverslip

• Cell media for infection

• Growth media appropriate for the cell line

• 0.4 % Trypan Blue Solution

• Lint-free wipes saturated with 70% isopropyl alcohol

• CO2 Incubator set at 37°C or 34°C, or other temperature as indicated

Procedure:

1. One day before infection, prepare 96 well dishes by seeding each well with Vero E6 cells in DMEM plus

7.5% fetal bovine serum, 4mM Glutamine, and antibiotics.

2. On the day of infection, make dilutions of virus samples in PBS.

3. Make a series of dilutions at 1:10 of the original virus sample. Fill the first tube with 2.0 mL PBS and the

subsequent tubes with 1.8mL.

4. Vortex the viral samples, then transfer 20 uL of the virus to the first tube, vortex, discard tip.

5. With a new tip, serial dilute subsequent tips transferring 200 uL.

Additions of virus dilutions to cells



1. Label the lid of a 96-well dish by drawing grid lines to delineate quadruplicates and number each grid to

correspond to the virus sample and label the rows of the plate for the dilution, which will be plated.

2. Include four (4) negative wells on each plate which will not be infected.

3. Remove all but 0.1 mL of media from each well by vacuum aspiration.

4. Starting from the most dilute sample, add 0.1 mL of virus dilution to each of the quadruplicate wells for

that dilution.

5. Infect four wells per dilution, working backward.

6. Allow the virus to absorb to the cells at 37°C for 2 hours.

7. After absorption, remove the virus inoculum. Start with the most dilute and work backward.

8. Add 0.5 mL infection medium to each well, being careful not to touch the wells with the pipette.

9. Place plates at 37°C and monitor CPE using the inverted microscope over a period of 1 to 4 weeks.

10. Record the number of positive and negative wells.

Protocol Changes:

Protocol Amendments: None

Protocol Deviations: None

Control Protocol

One control test was conducted without the SKYE unit in the testing chamber. Control samples were taken at

the corresponding sample time used for the challenge trial. Nebulization of viral media and collection methods

were the same for the control as the viral challenge. Control testing was used for the comparative baseline to

assess the viral reduction when the SKYE device was operated in the challenge trial to enable net reduction

calculations to be made. During the control test, four low volume fans were operated in each corner of the

testing chamber to ensure homogenous mixing of the air. During the control, temperature and relative

humidity were monitored. Prior to running the viral challenges temperature and humidity were confirmed to

be in relative range to the control ±5%.



Aerosolization of Viral Media:

The control samples were performed in the same manner as the viral test regarding the time points and

collection rate. A viral stock of SARS-CoV-2 USA-CA1/2020 with a concentration of 6.32 X 106 TCID50/mL was

used for this experiment.

Study Results RESULTS:

When tested against SARS-CoV-2 USA-CA1/2020 virus, the SKYE unit showed a reduction of active pathogens

after 30 minutes of continued operation resulting in a loss greater than 1.20 x 102 TCDI50/mL. This is indicative

of a 99.99% net reduction of collectable virus in the air after 30 minutes of operation.

**As it pertains to data represented herein, the value of 1.2E+02 indicates a titer that is lower than the

specified limit of quantitation. The limit of quantitation for this assay is 1.2E+02.

***As it pertains to data represented herein; the percentage error equates to an average of ±5% of the final

concentration.



Conclusion:

The Air+Health SKYE air purifier demonstrated the ability to reduce the concentration of the active pathogen,

SARS-CoV-2 USA-CA1/2020 from the air. During the test scenario, collectable active SARS-CoV-2 in the air was

reduced by 99.99% after 30 minutes of operation in the sealed testing environment. After 30 minutes of

operation, there was a loss greater than 1.20 X 102 TCDI50/mL indicating a titer that is lower than the specified

limit of quantification. The SKYE unit showed the ability to reduce collectable pathogen in the air below the

lower limits of detection faster than natural loss rates. As the test was designed to observe aerosol functions, it

is unknown if any active pathogen remained on the surface areas inside the SKYE unit or on the testing

chamber walls.

Effort was made to simulate a real-life environment in the chamber while taking into consideration the special

precautions needed when working with a Biosafety Level 3 Pathogen. Taking into consideration the starting

concentration of active SARS-CoV-2 virus, the volume aerosolized and the volume inoculated, one could

assume that the likelihood of entering an environment with this quantity of pathogen in a real-life circumstance

to be unlikely. Furthermore, when aerosolizing and collecting said pathogens, there are variables that cannot

be fully accounted for, namely, placement of pathogen, collection volume, collection points, drop rate, surface

saturation, viral destruction on collection, viral destruction on nebulization and possibly others. Every effort

was made to address these constraints with the design and execution of the trials.



Disclaimer The Innovative Bioanalysis, Inc. ("Innovative Bioanalysis") laboratory is not certified or licensed by the United

States Environmental Protection Agency and makes no equipment emissions claims pertaining to ozone or

byproduct of any Field Controls, LLC device. Innovative Bioanalysis, Inc. makes no claims to the overall efficacy

of any SKYE. The experiment results are solely applicable to the device used in the trial. The results are only

representative of the experiment design described in this report. Innovative Bioanalysis, Inc. makes no claims

as to the reproducibility of the experiment results given the possible variation of experiment results even with

an identical test environment, viral strain, collection method, inoculation, nebulization, viral media, cell type,

and culture procedure. Innovative Bioanalysis, Inc. makes no claims to third parties and takes no responsibility

for any consequences arising out of the use of, or reliance on, the experiment results by third parties.

_________________________________________ _______________________

Dana Yee M.D Date

Clinical Pathologist and Medical Director, Innovative Bioanalysis, Inc.

__________________________________________ _______________________

Sam Kabbani, MS, BS, MT(ASCP), CLS Date

Chief Scientific Officer, Innovative Bioanalysis, Inc.

__________________________________________ _______________________

Albert Brockman Date

Chief Biosafety Officer, Innovative Bioanalysis, Inc.

__________________________________________ _______________________

Kevin Noble Date

Laboratory Director, Innovative Bioanalysis, Inc.


7/3/2021

7/3/2021

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7/5/2021

efficacy of the field controls skye unit against

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